View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by HAL-INERIS Evaluation of an in vitro hsp 70 induction test for toxicity assessment of complex mixtures : comparaison with chemical analyses and ecotoxicity tests Selim Ait-Aissa, Pascal Pandard, H´el`ene Magaud, Andr´e-Patrick Arrigo, Eric Thybaud, Jean-Marc Porcher To cite this version: Selim Ait-Aissa, Pascal Pandard, H´el`ene Magaud, Andr´e-Patrick Arrigo, Eric Thybaud, et al.. Evaluation of an in vitro hsp 70 induction test for toxicity assessment of complex mixtures : comparaison with chemical analyses and ecotoxicity tests. Ecotoxicology and Environmental Safety, Elsevier, 2003, 54 (1), pp.92-104. <ineris-00961873> HAL Id: ineris-00961873 https://hal-ineris.ccsd.cnrs.fr/ineris-00961873 Submitted on 20 Mar 2014 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destin´ee au d´epˆot et `a la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publi´es ou non, lished or not. The documents may come from ´emanant des ´etablissements d’enseignement et de teaching and research institutions in France or recherche fran¸cais ou ´etrangers, des laboratoires abroad, or from public or private research centers. publics ou priv´es. 1 Evaluation of an in vitro hsp70 induction test for complex mixtures toxicity assessment: comparison with chemistry and ecotoxicity tests Sélim Aït-Aïssa,1* Pascal Pandard, 1 Hélène Magaud, 1 André-Patrick Arrigo, 2 Eric Thybaud1 and Jean-Marc Porcher1 1Ecotoxicological Risks Assessment Unit, INERIS, BP2, F-60550 Verneuil-en-Halatte, France 2Laboratory of Oxidative stress, Chaperones and Apoptosis, CNRS UMR 5534, University of Lyon-I, 69622 Villeurbanne Cedex, France Running title: hsp70 induction by industrial wastes eluates * To whom correspondence should be addressed. Fax:+33-344556767. E-mail address: selim.ait- [email protected]. 2 ABSTRACT The aim of this study was to assess the potential of a human cell line containing the hsp70 promoter linked to the chloramphenicol acetyl transferase reporter gene, to evaluate toxic potential of complex mixtures. Cells were exposed to eluates of industrial wastes and the cellular responses were compared to the metal contents of the samples and to standardised aquatic (microalgae growth inhibition, daphnia immobilisation, bacterial luminescence inhibition, Ceriodaphnia dubia reproduction inhibition) and terrestrial (earthworms lethality, plant growth inhibition) tests. The hsp70 promoter was significantly induced by 11 out of 14 samples, with different dose-response patterns. Significant correlation of in vitro induction potency with aquatic ecotoxicity, especially with chronic tests, and with the metal contents of the samples were observed. Our study provides new information on the relevance of hsp70 gene induction as a criterion of toxicity and suggests its usefulness for the detection of toxicity associated to metallic pollution in complex mixtures. Key Words: industrial wastes; heat shock protein; reporter gene; cellular stress; ecotoxicity tests; dose-response modelling; multivariate comparison. 3 INTRODUCTION Hazard evaluation of environmental complex mixtures consists in the determination of their intrinsic toxicity towards living systems and thus requires information given by both chemical analysis and toxicity testing. It has now become evident that chemical analysis is not sufficient to achieve hazard assessment of environmental samples, since it is restricted to some specific classes of toxic compounds, for which analytical methods are available and toxicity is known. In addition, chemical analysis do not inform neither on the available fraction for living organisms nor on the potential joint effects of the different contaminants. Hence, a toxicological evaluation using bioassays has to be done in order to determine the actual hazardous properties of a given sample. The potential of in vitro approach using cell tissues for assessing the harmful effects of chemicals on living systems has recently attracted much attention in ecotoxicology (reviewed by Walker, 1998; Wells et al., 1998). An emerging trend concerns the in vitro use of biochemical and molecular biomarkers for toxicity testing and the measurements of cellular responses related to the mechanisms of toxicity (Bierkens et al., 1998; Walker, 1998; Wells et al., 1998). In particular, cellular assays using reporter gene coupled to stress inducible gene promoters have gained acceptance as valuable tools for toxic potential evaluation. They are generally sensitive and specific, and their relative easy use and cost effectiveness allow their use in screening programs. At present, the application of receptor-mediated gene responses to environmental mixtures has been well studied with regard to the detection and quantification of specific contaminants such as dioxin-like compounds (Anderson et al., 1995; Kerr et al., 1999) or xenoestrogens (Zacharewski, 4 1997; Balaguer et al., 1999). The validity of the biological response has been achieved by correlating reporter gene induction with chemical analysis of the samples (Anderson et al., 1995; Vincent et al., 1997; Kerr et al., 1999). Besides, the application of cellular damages-mediated reporter genes, such as those involved in response to heat shock, oxidative stress and/or DNA alteration, has been less extensively characterised for toxicity assessment of environmental mixtures, although several studies have reported their activation by environmentally realistic pollutant levels (Fischbach et al., 1993; Guven et al., 1994; Van Dyk et al., 1994; Jones et al., 1996) or environmental samples (Vincent et al., 1997; Power et al., 1998; Hamers et al., 2000). These cellular responses are activated by stressors that alter vital cellular functions (i.e. proteins or DNA alteration), and are generally associated to early (cyto)toxic events (Goering et al., 1993; Neuhaus-Steinmetz and Rensing, 1997; Aït-Aïssa et al., 2000). Thus, they may represent potential predictors of toxic effects at higher levels of biological organisation. However, to our knowledge, no study compared such in vitro stress gene induction assays with in vivo (eco)toxicity effects. Unlike several in vitro cytotoxicity tests using primary or continuous cell cultures which have been shown to correlate to in vivo chemicals toxicity or ecotoxicity assays (Saito et al., 1993; Segner and Schüürmann, 1997; Gagné and Blaise, 1998), the predictive information provided by cellular reporter gene tests is left unexplored. In this paper, we address the use of heat shock gene (hsp70) induction in response to exposure to complex mixtures in a stably transfected cellular model. The heat shock response is characterised by the induction of a set of stress proteins, namely called heat shock proteins (HSPs). Under physiological conditions, HSPs act as molecular chaperons and ensure cellular protein homeostasis and cell protection. After cellular aggression, the release of the heat shock transcription factor (HSF) from its binding to HSP70 in the cytoplasm, allows formation of an 5 HSF homotrimer which migrates to the nucleus and binds the heat shock elements (HSE) in the promoter region of hsp genes, thus leading to the transcription activation of these genes. It is know admitted that the stressor-induced generation of misfolded proteins constitutes the trigger signal that up regulates the heat shock response (Hightower, 1991), and a number of environmental pollutants were shown to induce HSPs synthesis in many organisms or cell tissues (Sanders and Martin, 1993), through different mechanisms of action involving cellular protein alterations (Voellmy, 1996). Due to their strong conservation through evolution, the wide diversity of their inducing agents and the relative sensitivity of their expression in comparison to conventional endpoints such as growth, survival or reproduction, the HSPs, and especially the major stress protein HSP70, have been proposed as sensitive markers of non-specific effects in environmental monitoring ( Sanders, 1993; Ryan and Hightower, 1996; Bierkens, 1998). In a previous study, we used HeLa cells carrying the human hsp70 promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene (Kretz-Remy and Arrigo, 1994) to screen toxic chemicals (Aït-Aïssa et al., 1999; Aït-Aïssa et al., 2000). Heavy metals, pesticides, and chlorophenols induced the hsp70 promoter in this model, whereas some organic chemicals were found negative even at cytotoxic doses. The authors concluded that although HSP70 should not be considered as a universal marker of toxicity, it might be useful for the detection of toxic effects generated by many different types of chemical contaminants in complex samples. The aim of the present study was (i) to evaluate the applicability of the established hsp70-reporter gene model to reveal biological activity in complex mixtures, and (ii) to assess its toxicological significance by investigating how this cellular test compares to a battery of ecotoxicity tests and to chemical analyses. For this purpose, HeLa-CAT cells were exposed to fourteen eluates of industrial wastes and the cellular responses were compared to the metallic contents of the samples and to 6 ecotoxicological effects, as measured by standardised aquatic and terrestrial tests performed in parallel on the wastes (Pandard, 2000). MATERIALS AND METHODS Samples description, preparation and chemical analysis The table 1 summarises the characteristics and the origins of the industrial wastes used in this study. Sampling, waste description and sample preparation have been detailed elsewhere (Pandard, 2000). Briefly, eluates of wastes were prepared by mixing the raw waste with distilled water with a liquid to solid (dry matter) ratio of ten, for 24 hrs under circular agitation at room temperature, according to the draft european standard (prEN12457-2). After settling, the supernatant was collected and filtered through a 100 µm size pore filter. The pH was then adjusted between 5.5 to 8.5 if pH value was out of this range. The samples were filtered again through a 0.45 µm filter and then used for toxicity testing and chemical analysis. All aquatic ecotoxicity tests were performed on the 0.45 µm filtered fraction except the Daphnia magna acute test (see below) which was performed on the 100 µm filtered fraction. For in vitro testing, a further filtration of the samples through 0.2 µm filters was performed in order to avoid any bacterial contamination of the cultures. For metal analysis, the 0.45 µm filtrates were analysed by inductively coupled plasma atomic emission spectroscopy (ICP/AES) according to standard procedures (NF EN ISO 11885, NF EN 1483 and NF T 90-043). 7 In vitro cellular stress test HeLa-hsp70-CAT cells were routinely cultured in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated foetal bovine serum (Gibco, France) and 1% antibiotics (Penicillin-Streptomycin) at 37°C in a humidified atmosphere containing 5% CO . 2 Cellular test conditions were the same as those previously described for chemical compounds testing (Aït-Aïssa et al., 2000). Briefly, subconfluent cells (about 80% confluency) grown in 24- wells culture plates were washed twice with pre-warmed PBS and exposed in triplicates to various concentrations of the samples, ranging from 0.1 to 90% v/v. Just before exposure, nine volumes of sample were mixed with one volume of ten fold concentrated phosphate buffer saline (PBS), giving the upper range concentration (90 %), and thereafter serially diluted in serum-free culture medium. The former step allowed a salt correction of the sample as well as a pH stabilisation in physiological range values, thus preventing any osmotic or pH stress to the cells at the highest concentrations. Preliminary experiments showed that a 24 h exposure of cells in PBS did not neither modify the cell viability nor activate significantly the stress promoter. After a 16 hr exposure to wastes eluates, the cultures were washed once with pre-warmed complete medium and allowed to recover for 24 hours in complete medium before harvesting. Analysis of intracellular CAT content was performed with a CAT ELISA kit (Boeringher Mannheim) according to the manufacturer's instructions. Results are expressed as the ratio between the amount of CAT detected and the total cellular protein content as determined by a modification of the Lowry's method (BioRad DC Protein Assay kit). Cytotoxicity was evaluated by the total protein assay and was expressed as percentage of control cultures. Samples were stored at 4°C in the dark and tested for cellular stress 3 days maximum after preparation. This storage time did not 8 influenced their toxic potential since we observed that a storage for two weeks did not diminish significantly their in vitro toxicity (not illustrated). Ecotoxicological tests The ecotoxicity tests (Table 2) were performed according to standard procedures. Aquatic tests were performed either on the 100µm (Daphnia magna test) or on the 0.45µm (C. dubia, P. subcapitata and V. fischeri tests) filtered fractions. For all three terrestrials tests, dilutions of raw wastes were carried out in artificial soil (20 % Kaolinite clay, 10% sphagnum peat finely ground, 70 % fine quartz sand with more than 50 % of particle size between 0.02 mm and 0.5 mm). Analysis of hsp70 induction data Hsp70 induction curves parameters, such as effective concentrations, maximal induction level and the rate of the induction, were calculated after modelling the observed in vitro patterns. Because hsp70 induction profiles were often balanced by a cytotoxic effect, thus leading to biphasic induction curves, the measured effects were modelled as follows. Firstly, cytotoxicity data, expressed in % of control cultures, were fitted to a logistic model according to equation (1) : A D (1) y D B concentration 1 EC 50 where y = (cyto)toxicity, expressed in % of control, A = no effect value (set to 1), B = parameter representative of the slope of the viability fall, EC = concentration leading to 50% (cyto)toxicity 50 compared to control and D = maximal effect value (set to 0 for 100% toxicity). The resulting